New member of the road crew tending the molecular highway

July 3, 2002

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By Barbara Berg

Drs. Fred van Leeuwen and Phil Gafken display a mass spectromer they used to characterize a chemical modification that may help organize the yeast genome into active and inactive regions.

Photo by Caren Brinkema

Tangled with DNA and clogged with pro- teins navigating biological soup, a cell's nucleus resembles a freeway jammed with motorists in search of destinations.

Molecular signposts along the chromosome highways likely direct proteins to their proper exits, where they act to switch genes on or off. Yet scientists have been challenged to learn the chemical nature of these route markers and the identity of the proteins that construct them.

Now, researchers in the Basic Sciences Division have identified a yeast protein likely to be a member of the road crew. Known as Dot1, the enzyme stamps a chemical roadblock on chromosomes, effectively diverting a family of proteins charged with shutting off genes. By steering these proteins away from the wrong destinations, Dot1 ultimately may help organize the genome into its proper active and inactive regions.

What's more, the researchers suspect that navigation systems like those mediated by Dot1 are likely to be discovered in other organisms including humans, as many cell types harbor analogous, though as yet uncharacterized, proteins.

Reported in the June 14 issue of Cell, the study was led by Dr. Fred van Leeuwen, postdoctoral fellow in Dr. Dan Gottschling's laboratory.

New way to think

Gottschling said the findings offer a new way to think about how cells - tiny compartments packed with molecules - achieve the remarkable precision and accuracy essential to many biological processes. Without such specificity in gene expression, for example, the wrong genes might get activated or repressed in response to certain stimuli, possibly leading to diseases like cancer.

Gene activation and repression are achieved by the binding of protein regulators to discrete spots on DNA, where they coax genes into active or inactive states. Such regulators bind to their intended sites presumably because they have properties that let them "stick" to some DNA sequences (high-affinity sites) more tightly than others.

"Most people think of specificity in these terms - that is, proteins don't bind promiscuously mainly because of this strong attraction to high-affinity sites," Gottschling said. "But in fact, regulatory proteins can have a weak attraction to other DNA sequences along the chromosome, yet they don't tend to bind there. What we think may prevent such nonspecific binding are chemical modifications on chromosomes that further decrease the attractiveness of these low-affinity sites. Dot1 makes one such modification.

Dot1, which stands for a disruptor of telomeric silencing, was identified in Gottschling's lab several years ago in a hunt for mutations that cause normally silent parts of the genome - regions where genes are rarely turned on - to become active. Telomeres, the ends of chromosomes, are one such silenced domain.

Silent regions of the genome are kept hushed in part by the binding of a group of silencing proteins. In mutant yeast cells that are missing Dot1, such silencing proteins move away from the telomeres to other parts of the genome, and silencing at telomeres is alleviated, van Leeuwen said.

"This causes the silencing proteins to be diluted out all over the genome, rather than concentrating at telomeres or other silent regions," he said.

In other words, without Dot1, parts of the chromosome that ordinarily would be weakly receptive to binding by silencing proteins become more attractive. This suggests that Dot1's role is to establish roadblocks that bar silencing proteins from these normally active regions.

Gottschling compared this hypothesis to a typical scenario in his former Chicago neighborhood.

"In Chicago, there are lots of pigeons, and they like to hang out on windowsills," he said. "People don't want pigeons on their windowsills for obvious reasons, so they put metal spikes on the sills to keep the birds away. The pigeons now flock to other, more attractive windows - those without spikes. In effect, Dot1 puts up spikes on the chromosome to keep silencing proteins away so they can bind where they should."

Clues to the chemical identity of these spikes came from a comparison of the sequence of Dot1 to other sequenced genes. A segment of Dot1 is similar to a family of other proteins known as methyltransferases, enzymes that attach chemical tags known as methyl groups to other molecules.

Van Leeuwen, working with Dr. Phillip Gafken, manager of the center's proteomics faculty and a co-author of the paper, discovered that Dot1 attaches methyl groups to a protein on the chromosome's backbone known as histone H3.

Family of proteins

While chromosomes are best known for their DNA composition, the organization, structure and even regulation of chromosomes depends on a family of proteins, the histones, that form a scaffold around which the DNA wraps.

Histones are known to undergo many chemical modifications, but none in the core region where Dot1 makes its mark - and none that cause such a global shift in binding of silencing proteins.

Gottschling and van Leeuwen postulate that Dot1's mark may do more than help to establish silencing at the right genomic locations. Silent or active states on chromosomes are typically inherited as a cell divides into two. The methyl groups added by Dot1 may act as a sort of a molecular string tied around a finger that provides the genetic memory enabling silenced or active states to be passed on to daughter cells.

If true, Dot1 would distinguish itself as a highway engineer that also provides easy-to-follow directions.

Perhaps the state Department of Transportation could learn a lesson from biology.

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